Thermal cycling device

ABSTRACT

Multi-layer devices suitable for thermal cycling processes. The devices are particularly suitable for performing polymerase chain reactions (PCR). One embodiment includes a first conducting layer, a second conducting layer adjacent to the first layer, and a third conducting layer adjacent to the second layer opposite the first layer. Insulating layers are positioned between said three conducting layers. Continuous channels are formed within the layers. The channels can be formed in either the conducting layer or the insulating layers, or both. Other embodiments include two conducting layers. At least one integral or separate temperature source may be provided to maintain the conducting layers at various desired temperatures.

RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 11/744,676 filed on May 4, 2007, which is acontinuation-in-part of U.S. patent application Ser. No. 10/906,546,filed on Feb. 24, 2005. The present invention also claims priority toU.S. provisional applications 60/547,036, filed Feb. 24, 2004,60/629,910, filed Nov. 22, 2004, and 60/745,550, filed May 5, 2006, thedisclosures of which are incorporated by reference herein.

TECHNICAL FIELD

The present invention relates generally to a thermal cycling devices,and more specifically, to a multi-layer thermal cycling devices suitablefor the life science, medical devices and biotechnology fields. Onesuitable application is to perform a polymerase chain reaction (PCR).

BACKGROUND

Various fields such as life science, medical devices and biotechnologyoften require thermal cycling for performing various reactions. One typeof reaction is the polymerase chain reaction (PCR) in which a biologicalsample such as a DNA fragment is replicated.

PCR has been the preferred method for replicating, or “amplifying”specific and singular nucleic acid constituents in an otherwise complexsample. Among the requirements of the PCR protocol is that the PCRsample, consisting of the biologic sample and PCR reagents, be exposedto three distinct temperatures for a specific length of time. Thetemperature and time of the exposure is optimized for the particularnucleic acid sequence desired. It is also a requirement that the PCRsample be exposed to the three temperatures multiple times. In thescientific vernacular, these two requirements are commonly referred toas thermal cycling.

Scientific endeavors such as the Human Genome Project and other similarefforts require that an extraordinary number of PCR reactions beconducted. The costs and time associated with the performance of PCR canbecome prohibitive for scientific studies that require thousands or evenmillions of PCR reactions. Cost reduction can be realized by reducingthe volumes of the materials required for a successful PCR reactionthrough miniaturization. Total PCR reaction time can be reduced bydecreasing the time required for the sample to be exposed to, andequilibrate at each of the temperature set points in the prescribed PCRprotocol.

Current art has relied on various means to achieve these goals. Eachmethod and device contains one or more of the following attributes thatprohibits rapid temperature changes in the PCR sample. The temperatureof one or more sources of thermal energy must be changed to establisheach temperature set point. The time required for this is determined bythermal conductivity of the device material which is inherently slowerthan the PCR sample itself. If constant temperature thermal sources areemployed, regional areas of the device equilibrate at the desiredtemperature set points but gradual temperature changes are establishedin the surrounding areas within the device due to the thermalconductivity properties of the materials. This results in a slow thermaltransition of the sample as it moves through the device. The time totraverse these transition temperature ranges serves to increase thetotal reaction time in comparison to methods that offer abrupt orinstantaneous changes. It also requires that the device be larger andtherefore does not provide the highest degree of miniaturization. Thus,the full cost benefit from reduced PCR sample volumes is not realized.

There is substantial development of devices and methods to facilitatethe temperature cycling, also referred to as thermal cycling. U.S. Pat.No. 6,337,435 TEMPERATURE CONTROL FOR MULTI-VESSEL REACTION APPARATUS byDaniel Chu, et al. describes an approach whereby a sample or samples ina multi-well container are subjected to temperature changes caused byphysical contact between the multi-well container and a heating device.The time required to perform the thermal cycling is largely determinedby thermal mass and other physical properties of the heating device andis therefore relatively slow.

The multi-well device is usually opened at the top to provide access tothe wells for the introduction and removal of the sample. This createsan environment for the undesirable evaporation of the sample. Theaforementioned patent, as well as others, strive to reduce this effectby implementing covers. The covers reduce evaporation but present anopportunity for undesirable condensation. This effect is often mitigatedby heating the cover but this then introduces a competing temperaturesource to the system, further increasing the total PCR reaction time.

There has been a significant effort within the biotechnology communityto create closed miniaturized devices in order to mitigate thedisadvantages of the open well/vessel systems. The advantages of aclosed miniaturized system would be to reduce sample evaporation, reducecondensation, and to reduce costs by using smaller sample and reagentvolumes. Exemplary of such miniaturized systems is U.S. Pat. No.6,284,525 MINIATURE REACTION CHAMBER AND DEVICES INCORPORATING SAME byRichard Mathies and Adam Woolley and U.S. Pat. No. 6,261,431 PROCESS FORMICROFABRICATION OF AN INTEGRATED PCR-CE DEVICE AND PRODUCTS PRODUCED BYTHE SAME by Richard Mathies, Peter Simpson, and Stephen Williams. Suchdevices are fabricated containing closed reaction chambers. Sample andreagent liquids flow into and out of these chambers through a network offluid channels. Heating elements such as resistive wire elements arefabricated within these chambers and provide the heating energy requiredto execute the PCR assay.

As with the larger and open system, the thermal properties of the deviceand the structural design primarily determine the reaction time.Although these systems are an improvement over the previously describedapproach, the thermal characteristics of such a structure is a limitingfactor. Also, fabricating, controlling, and monitoring of the in situheating elements is complicated and adds appreciably to the cost of thedevice.

Another method that attempts to accelerate the PCR process is describedin U.S. Pat. No. 6,180,372 METHOD AND DEVICES FOR EXTREMELY FAST DNAREPLICATION BY POLYMERASE CHAIN REACTIONS (PCR) by Jochen Fanzen. Inthis embodiment, a two dimensional network of microfluidic channels iscontained within two temperature heating/cooling elements. Themicrochannel device is exposed to rapid temperature changes by changesin the heating/cooling elements above and below the device. This systemis very efficient in the energy transfer between the heating/coolingelements due to complete physical contact with the elements. However,the total PCR reaction time is still limited by the ability to changetemperature within the heating/elements and within the microchannelmaterial.

The above-mentioned devices and methods address the issue of thermalcycling for PCR by various configurations of heating elements in anattempt optimize the energy transfer from the elements to the device andeventually the PCR sample contained there in. All of these methods anddevices are limited by their ability to transfer thermal energy throughthe device and into the sample. Further limitations are present when theheating elements themselves must also change temperature in order toexpose the PCR sample to each desired temperature set point in the PCRprotocol.

It is therefore desirable to provide a thermal cycling device thatreduces the thermal limitations of prior known devices as well asreducing evaporation, condensation and cost so that a device for rapidlyperforming thermal cycling is provided.

SUMMARY

The present invention provides a method and apparatus for improvedthermal cycling performance.

In one aspect of the invention, an apparatus includes a first layer, asecond layer proximate to the first layer, a third layer proximate tothe second layer opposite the first layer, and a continuous channel. Thecontinuous channel is formed within the first layer, second layer andthird layer. The continuous channel has a plurality of cycle segments.Each of the cycle segments comprises a first portion disposed within thefirst layer, a second portion disposed within the second layer, and athird portion disposed within the third layer.

In a further aspect of the invention, a device for performing a reactioncomprises a first heating means, a first thermally conductive layerthermally coupled to the first heating means, a first insulating layerproximate the first thermally conductive layer, a second thermallyconductive layer directly adjacent to the first insulating layer and asecond insulating layer directly adjacent to the second thermallyconductive layer. A third thermally conductive layer is disposedproximate to the second layer opposite the first layer. A continuouschannel is formed through the first thermally conductive layer, thefirst insulating layer, the second thermally conductive layer, thesecond insulating layer, and the third thermally conductive layer. Thecontinuous channel has a plurality of cycle segments, each of the cyclesegments comprising a first portion disposed within the first thermallyconductive layer, a second portion disposed within the third thermallyconductive layer, and a third portion disposed within the secondthermally conductive layer.

Additional embodiments of the invention include two and three layerdevices which include the fluid channels in one or more of theinsulating layers. Other embodiments include fluid flow channels which“fold back” from one-side of the device to the other in the same layeror temperature strata. Further embodiments include tabbed connectors toone or more of the thermal conductive layers in order for heating orheat sinking purposes.

In yet another aspect of the invention, a method of performing areaction comprises introducing a sample into a device having a firstlayer, a second layer, and a third layer performing a first portion of acycle in a first layer at a first temperature, moving the sample to thethird layer, thereafter performing a second portion of the cycle in thethird layer at a second temperature lower than the first temperature,moving the sample to the second layer, thereafter performing a thirdportion of a cycle in the second layer at a third temperature betweenthe first temperature and the second temperature, and repeatedlyperforming the first portion, second portion and third portion for apredetermined number of cycles to perform the reaction.

Other method embodiments include two and three layer devices which movethe samples through channels in one or more of the insulating layers andother devices wherein the channels “fold back” from one side of thedevice to the other in the same layer or temperature strata.

One advantage of the invention is that only two sources of thermalenergy are required to perform a polymerase chain reaction that usesthree different temperatures. Advantageously, because the upper andlower layers are maintained at a temperature, delays due to thermalcycling of the individual layers are not present. This accelerates theentire process compared to the existing art.

Other advantages and features of the present invention will becomeapparent when viewed in light of the detailed description of thepreferred embodiment when taken in conjunction with the attacheddrawings and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Various features and advantages of the present invention will becomereadily apparent upon reading the following detailed description uponreference to the attached drawings, in which:

FIG. 1 is a perspective view of first embodiment of the thermal cyclingdevice according to one embodiment of the present invention.

FIG. 2 is an exploded view of the thermal cycling device of FIG. 1.

FIG. 3 is a partially broken away cross-sectional of the thermal cyclingdevice illustrated in FIGS. 1 and 2.

FIG. 4 is a perspective view of the channel of the device of FIGS. 1through 3.

FIG. 5 is an exploded view of a second embodiment of the invention.

FIGS. 6A-6D are cross-sectional views of the device of FIG. 5.

FIG. 7 is a cross-sectional view of a third embodiment of a thermalcycling device of the present invention.

FIG. 8 is a cross-sectional view of the fourth embodiment of the thermalcycling device according to the present invention.

FIG. 9 is a perspective view of a heater formed according to the presentinvention.

FIG. 10A is a plot of bulk resistivity versus thickness for a heaterformed according to FIG. 9.

FIG. 10B is a plot of bulk resistivity versus n-type carrier doping forthe heater illustrated in FIG. 9.

FIG. 11 is a cross-sectional view of a fifth embodiment of the presentinvention.

FIG. 12 is a schematic view of a PCR analyzer and kits for the same.

FIG. 13 is a cross-sectional view of a channel that uses bubbles toimprove the thermal cycling.

FIG. 14 is a cross-sectional view of a channel portion having an LED anda photodiode therein.

FIG. 15 is a schematic view of a device according to the presentinvention.

FIG. 16 is a cross-sectional view of a sixth embodiment of theinvention.

FIG. 17 is a cross-sectional view of a seventh embodiment.

FIG. 18 is a perspective view of a fluidic test device formed accordingto the present invention.

FIG. 19 is an exploded view of the device of FIG. 18.

FIG. 20 schematically illustrates a three-temperature device in whichfluid channels are formed in an insulating layer.

FIGS. 21A and 21B illustrate a two-temperature device in which fluidchannels are formed in an insulating layer.

FIG. 22 schematically illustrates another three-temperature device inwhich fluid channels are positioned in both sides of the extensionlayer.

FIG. 23 schematically illustrates another two-temperature device inwhich fluid channels are positioned in both sides of theannealing/extension layer.

FIG. 24 schematically illustrates another three-temperature device inaccordance with the present invention.

FIGS. 25A and 25B illustrate another two-temperature device inaccordance with the present invention.

FIG. 26 illustrates a thermal cycling device with tabbed connectors inthe conducting denaturation layer.

FIG. 27 is an exploded view of the thermal cycling device of FIG. 26.

FIG. 28 schematically illustrates still another thermal cycling devicein accordance with the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the following figures the same reference numerals will be used toidentify the same components. It should be noted that various examplesincluding reagents, time, materials, and channel configurations are setforth below by way of example and are not meant to be limiting. Thepresent invention is also described with respect to a polymerase chainreaction (PCR). However, various biological and chemical fields maybenefit by the use of the present invention. It should also be notedthat although the device is suitable for thermal cycling, a heatingdevice may not be integral to the apparatus. That is, the heating devicemay be an external device coupled to the apparatus.

Referring now to FIG. 1, a thermal cycling device 10 includes a fluidicdevice 12, a first temperature source 14, and a second temperaturesource 16. The fluidic device, as illustrated, is formed of a firstthermally conductive layer 18, a second thermally conductive layer 20,and a third thermally conductive layer 22. The layers may be planar andrectangular in shape. The layers may also be separate layers or strataof the same thermally conductive layer. That is, in a device the layersmay be areas where the temperature is the same across the device. Thematerials of the device may be the same or different, with or withoutinsulation as will be further described below. Various types ofmaterials having high thermal conductivity may be used for the presentinvention. The high thermal conductivity causes the temperature acrossthe layer to become isothermal. For example, metals such as steel onaluminum and semiconductors such as silicon are most preferredmaterials. However, other materials such as glass and various types ofcomposite plastics may be used for the thermally conductive material.Each layer may be formed of a different or the same materials. Thelayers are proximate to each other. That is, the layers may be directlyadjacent, adjacent, or have heaters, insulators or other structuresdisposed therebetween.

Insulating layers may be used to at least partially thermally isolatethe various layers. A first insulator 24 is disposed between the firstthermally conductive layer 18 and the second thermally conductive layer20. A second insulator 26 is disposed between the second thermallyconductive layer 20 and the third thermally conductive layer 22. Thefirst insulator 24 and second insulator 26 may be formed as a planarlayer between the thermally conductive layers. Of course, otherembodiments of such an insulator would be evident to those skilled inthe art. The first insulator 24 and second insulator 26 are preferablyfabricated from materials with low thermal conductive properties, suchas polypropylene, polycarbonate, polyethylene or polyimide. As is bestshown in FIG. 2, the insulators 24, 26 include various ports 28therethrough so that fluid may be transferred between the thermallyconductive layers. These layers may be drilled or deep reactive ionetched. The insulators 24, 26 act as a thermal buffer between the layersso that three temperatures may be maintained using two temperaturesources. That is, each of the layers rapidly equilibrate and thus areisothermal. By providing isothermal layers the temperature transfer isextremely rapid. The two limiting factors in a PCR process will be thethermal absorption of the sample and the enzymatics of the chemistry.Thus, PCR may be performed as fast as nature will allow.

Ports 28 may be formed in various manners including drilling or laserablation. When in a layer form, the first insulator has a top surface 30and a bottom surface 32. The top surface 30 is adjacent to the bottomsurface 34 of the first conductive layer 18. The bottom surface 32 ofthe insulator 24 is directly adjacent to the top surface 38 of thermallyconductive layer 20. The bottom surface 40 of second thermallyconductive layer 20 is directly adjacent to the top surface 42 of thesecond insulating layer 26. The bottom surface 44 of insulator 26 isdirectly adjacent to the top surface 46 of third thermally conductivelayer 22. The top surface 36 of first thermally conductive layer 18 hasan inlet port 50 therein. Inlet port 50 may be used to provide a fluidsample to the fluidic device 12. An outlet port 52 is used to remove thefluid sample from the fluidic device 12. The layers may be coupledtogether using a thin layer of epoxy or other adhesive. Alignment guidesor pins may be used in alignment of the device during manufacturing.

As will be further described below, various types and positions oftemperature sources 14, 16 may be used. Separate devices such as platensor integral devices may be used. As is illustrated in FIG. 3, the firsttemperature source 14 and the second temperature source 16 are directlyadjacent to the top surface 36 of the thermally conductive layer 18 andthe bottom surface 48 of third layer 22, respectively. The layeredtemperature sources 14, 16 are thus integral to the device 12 in FIG. 3.

An outlet port 52 is illustrated through third thermally conductivelayer 22. The outlet port allows the fluid sample to exit the device. Acontinuous channel 54 is illustrated through the device. The continuouschannel may be broken into various cycles that have portionscorresponding to each layer of the device. In a PCR environment, threedifferent temperatures are desired. Exposure to three differenttemperatures forms one amplification cycle of the PCR reaction.

The continuous channel includes an entry portion 56, a first cycleportion 58 disposed within the first thermally conductive layer 18, anda connection portion 60 that couples the fluid sample from the firstthermally conductive layer 18 to the third thermally conductive layer22. The third thermally conductive layer 22 has a second cycle portion62 therein and is thus exposed to a second temperature less than thefirst temperature of the first thermally conductive layer. The secondcycle portion 62 is fluidically coupled to a second connection portion64 that couples the fluid from the second cycle portion 62 to the thirdcycle portion 66. The third cycle portion 66 is disposed within thesecond thermally conductive layer 20. As will be described below,various numbers of cycles through the first portion 58, second portion62, and third portion 66 may be performed depending on the variousparameters and desired amplification in a PCR process. Once thepredetermined number of cycles through the device is performed, thefluid sample leaves the device through the third layer 22 through anexit portion 68. Exit portion 68 is performed when the fluid passesthrough the desired number of cycles formed in the fluidic device 12.The lengths and cross-sectional areas of the first portion 58, secondportion 62, and third portion 66 are sized so that the fluid sample ineach pathway is exposed to the temperature for a predetermined amount oftime. The size of the pathway may be changed by varying thecross-sectional area and the length. The dwell time is proportional tolength and inversely proportional to channel cross-sectional area. Thefluid inlet 50 and fluid outlet 52 may be positioned in various layers.The position may be dictated as a matter of convenience depending on theconfiguration of the equipment to which the fluidic device is attached.

The first temperature source 14 and second temperature source 16 may beformed of various types of materials such as electric thin film devices,resistive wire devices, platens or other sources of constanttemperature. In the embodiment of FIG. 3, the temperature of the upperlayer is maintained at a first temperature with the first temperaturesource 14 and the third layer 22 is maintained at a second temperatureless than the first temperature by the second temperature source 16. Theupper surface of the first layer and the lower surface of the thirdlayer 22 are illustrated as being contacted by the temperature sources14, 16, respectively. It should be noted that the edge or edges of oneor both of the layers may also be contacted by the temperature sources.Preferably, the temperature sources are regulated. Various feedbackdevices such as thermistors or thermocouples may be used to providefeedback to a controller. The second thermally conductive layer 20absorbs heat from each of the layers in a controlled manner through theinsulators 24, 26. That is, the amount of absorption may be controlledby controlling the amount of thermal transfer through the insulatinglayers by thickness or material choice. The temperature of the secondlayer is maintained at a third predetermined temperature between thefirst temperature and the second temperature. It may take a finiteamount of time for the entire device to reach equilibrium. Theconductive layers are preferably highly thermally conductive and thusare isothermal. After an equilibrium is reached, fluid or fluid samplesmay be passed through and may be exposed to each of the different layersin each of the different cycles through the continuous channel 54.Because the layers 18, 20 and 22 may be highly thermally conductive, lowor nearly no temperature differentials may be supported within thethermally conductive layers. Because the insulating layers 24 and 26 areof low thermal conductivity materials, high temperature differentialsmay be supported within these layers. The second thermally conductivelayer 20 or extension layer is warmed by the influx of heat from thefirst conductive layer 18 or denaturation layer through the upperinsulating layer. The second thermally conductive layer 20 or extensionlayer is cooled by convection, radiation and conduction from its edges,efflux through the lower insulating layer and by the influx of liquidfrom the annealing layers. Its steady state temperature is the result ofbalancing these competing energy fluxes. Edge losses can be madenegligible by the design of a holder for the device. The thermalresistance of the insulating layers is selected with two considerations.When no liquid is flowing, the steady state temperature of the extensionlayer will be set by the relative thermal resistance of the twoinsulating layers. When flow is present, the steady state temperature ofthe extension layer will drop by an amount that depends on how muchenergy is required to heat the influx of liquid. This drop must bewithin the range of extension temperature that PCR can allow. Theconstraint sets an upper bound on the thermal resistance of theinsulating layer and, therefore, a lower bound on the energy that mustbe supplied to the device. In general, the energy that must be suppliedto the device consists of two terms, both proportional to the differencebetween the denaturation and annealing temperatures. The first term isthe part that establishes the steady state heat flux in the absence ofliquid and second term is due to the overhead of heating liquid enteringthe denaturation and extension layers. Ignoring edge effects, all of theenergy supplied to the device may be removed with suitable heat sinking.

The fluid sample has a rate of absorption which is one limiting factorto the speed of the process. The enzymes of the chemistry may alsolimit. Contrary to the prior art, the present device is limited by onlythese two constraints. The geometry of other art limits the speed ofthose processes. Typically, long pathways exist to achieve thermalisolation in such devices.

Once the fluid is placed within the continuous channel 54, various meansfor moving the sample may be provided such as capillary forces, vacuum,a pressure source or other means. The movement means is illustrated as afirst movement means 70 and a second movement means 72. One or bothmeans 70, 72 may be present.

The size, materials and fabrication techniques employed in thisembodiment may be selected to most effectively address the needs of thespecific application. For example, molding, machining, casting,extruding and various types of metals and plastics may be used for thefluidic device 12. Also, various types of heaters may also be employed.Micro-electrical mechanical systems (MEMS) may also be used to fabricatesuch a device. Photolithographic processes may result in the creation ofmultiple layers of dissimilar materials that contain the appropriatepathways, entry and exit ports, and ports through the device. Also, onefluidic device is illustrated. It should be noted that multiple fluidicdevices may be employed adjacent to each other in a multiple reactiondevice. That is, several reactions may take place simultaneously orsequentially within the device. The fluid path may be several individualchannels connected in parallel. This may be useful for thermal cyclinglarge volumes of fluid more rapidly. The device may also be disposablein a one-time use application, or may be washed and reused.

Referring now to FIG. 4, a ten cycle device 12′ is illustrated withoutthe layers and only showing the continuous channel 54′. In thisembodiment the first portion 58′ of the continuous channel 54 isillustrated. The connection portion 60′ connecting the first portion tothe second portion 62′ is also shown. After leaving the second portion62′ the fluid enters the second connection portion 64′. Fluid thenenters the third portion 66′ before entering the third connectionportion 68′. Thus, the portions 58 through 68 illustrate one cyclethrough the device. In a PCR device the first portion is a denaturingportion. The first portion 58′ is a denaturing portion. The secondportion 62′ is an annealing portion, and the third portion 66′ performsan extension portion. In this illustration the cycle repeats ten timesuntil the sample reaches the outlet 52′ which is illustrated in anupward position and thus would correspond to the thermally conductivelayer 18. Various positions of the inlet port 50 and the outlet port 52may be evident to those skilled in the art. In this embodiment, thechannels may be formed of a tubular structure and overmolded to form aunitary device. It should also be noted that the tubular device may beovermolded in the three thermally conductive layers described in FIGS.1-3.

Referring now to FIG. 5, an exploded view of a second embodiment 74 isillustrated. In this embodiment the fluid inlet port 50″ is disposed inthe third layer 22″. The fluid inlet port 50″ extends through the secondinsulator 26″, the second thermally conductive layer 20″, the firstinsulator 24″, and the first thermally conductive layer 18″. An initialdenaturing channel 76 is formed within the thermally conductive layer18″. In this constructed embodiment, the initial denaturing channel 76was 6.5 mm. The channel portions set forth in this embodiment aretriangular in cross-section. The channels were formed using orientationdependent etching using photolithographic techniques. A plurality offirst cycle portions 58″ are illustrated within the first thermallyconductive layer 18″. The first cycle portions 58″ is fluidicallycoupled to the initial denaturing channel 76. In this embodiment aplurality of connection portions 60″ are illustrated. These portions maybe drilled or deep reactive ion etched. This embodiment has alongitudinal axis X and a lateral axis Y. Each of the first portions58″, second portions 62″, and third portions 66″ are laterally disposedexcept for a slight L-shaped variation on the first portion 58″. In thisembodiment, fluid flows generally from the thermally conductive layer18″ to the third layer 22″ through the first connection portion 60″.Fluid then enters the extension plate which is the second layer 20″.Fluid thus passes laterally outwardly from the center of the thirdportion 66″ where it then passes up to one of the rows of first portions58″. As will be illustrated below in FIG. 6, the fluid passes throughalternate sides of the device for each cycle. In this embodiment thechannels are etched in the lower surface 34″ of the first thermallyconductive layer 18″, in the top surface 38″ of the second layer 20″,and in the top surface 46″ of the bottom layer 22″. The first insulator24″ and the second insulator 26″ help define the fluid passage channels.In the constructed embodiment, 30 PCR cycles are performed with a 6.5 mminitial denaturing channel, wherein the next and subsequent denaturingchannels are 1 mm long. The annealing channels or third portions 66″ arealso 1 mm long. The extension channels or the second portions 62″ are 2mm in length. It should be noted that an epoxy or other adhesive may beused to couple the conductive and insulative layers together.

The device may be fabricated using techniques borrowed from thermal inkjet cartridges and integrated chip manufacturing. The device may beformed of silicon and later diced after being bonded together. Alignmentfeatures may be incorporated into the device to allow various fixturesto assist in assembling the device.

As is best illustrated in FIGS. 6A-6C, fluid flow in a first cycleportion 58 a″ extends laterally inwardly and through the firstconnection portion 60 a″ where it enters second cycle portion 62 a″. Thefluid flow within the second cycle portion 62 a″ is also laterallyinwardly. Fluid then flows through one of the second connection portions64 a″ laterally outwardly through a third cycle portion 66 a″. Thus, thefluid at this point is on a laterally opposite side of the fluidicdevice 12″ than when the fluid entered within the first cycle portion 58a″. The fluid then enters a third connection portion 68 a″ where itenters another first portion 58 b″ on the opposite side of the devicefrom the first cycle portion and travels laterally inwardly in adirection opposite to that from the first cycle. When the fluid entersthe first portion 58 b″, a second cycle is initiated. Fluid then flowsin an opposite direction to those described above in that within thesecond portion 62 b″ the fluid flows laterally inwardly from the leftside to the middle of the device. When transitioning between the variousthermally conductive layers the fluid passes through various ports 28″in each of the first insulator 24″ and second insulator 26″. Also, inthis embodiment the outlet port 52″ is through the third thermallyconductive layer 22″. As is best show in FIG. 3, the third cycle beginsin the first cycle portion 58 c″.

Referring now to FIG. 7, a third embodiment 80 of fluidic device 12′″ isillustrated. In this embodiment, a glass layer 82 is provided upon thefirst thermally conductive layer 18′″. The glass layer 82 allowsfluorescent or other optical detection therethrough. The glass layer 82is preferably transparent at the detection wavelength and thus theamount of DNA within the first portions 58′″ may be monitored. Also, inthis embodiment a heater layer 84 is employed between the firstthermally conductive layer 18′″ and the first insulator 24″. A secondpolysilicon heater layer 86 may also be used in place of the heaterillustrated in FIGS. 1-4. One suitable type of heater is a polysiliconheater. Various polysilicon coatings are commonly used for inkjetdevices. Although the heater is illustrated as a separate layer dopingmay be used to form the layer. Also, the layer may be formedepitaxially. As is described elsewhere the heaters may contact theedges, or faces of the layers.

Referring now to FIG. 8, a fourth embodiment 90 is illustrated using anelectrical “joule” heating device. A first electrical source 92 and asecond electrical source 94 are coupled to an joule heater 96 and 98,respectively. That is, the joule heater 96 may be disposed on the uppersurface of the thermally conductive layer 18″″. The second joule heater98 is disposed on the bottom surface of the third layer 22″″. In apractical embodiment a thermocouple, thermistor or other sensing devicemay be required to control the temperature. The device may be externalto or integrated with the thermal cycling device in various ways knownto those skilled in the art.

As is best shown in FIG. 9, the current may pass through the deviceusing a pair of contacts 100, 102. The electrical contact may be made onthe surface or at the edges of the silicon by various commonly employedmeans. By providing the potential difference between the contacts 100,102, a predetermined amount of current may pass therethrough and cause aspecific amount of heating. The conductivity required may becharacterized by the surface resistance of the material q_(s) with unitsof ohms per square or simply ohms. q_(s) is the bulk resistivity q,divided by the thickness of the conducting layer. In the case of bulkconduction, the entire thickness of the silicon layer may be used. Inthe simplest situation contact is made across the width of the device ateach end as is shown in FIG. 9. Current flows through the surface layeror bulk, depending on the configuration. Power is dissipated per unitarea as V²/q_(s)L² and the current per unit of width is V/q_(s)L. For adevice where L is 1 centimeter and q_(s) is 10 ohms per square, applyingten volts will result in dissipation of 10 watts per square centimeterat a current density of 1 ampere per centimeter of width. Any particulardevice may require more but usually much less than this amount ofheating.

Referring now to FIGS. 10A and 10B, FIG. 10A shows the bulk resistivityrequired to achieve 10 ohms per square for a conducting layer ofspecified thickness. For example, a 500 micron (0.55 mm) thick siliconwafer will have a surface resistance of 10 ohms per square if the bulkresistivity is 0.5 ohms-cm. The same surface resistance will result if a10 micron thick layer having a resistivity of 0.01 ohms-cm were createdon the surface of the silicon through metal plating, ion implantation orion diffusion techniques. If doping or implantation is to be used, FIG.10B indicates the levels of n-type carriers needed to achieve therequired bulk resistivity. P-type doping may also be used.

Referring now to FIG. 11, if bulk joule heating is employed in a siliconlayer, contacts may be provided on the lateral (or longitudinal) edgesof the device. As is illustrated, contacts 104 and 106 are provided tothe first thermally conductive layer 18″″. A second set of contacts 108,110 are coupled to the third layer 22″″. It should be noted that theelectrical contacts may be integral to the device or separate from it.Thus, current may flow in bulk through the device and provide aspecified joule heating. Such configurations may also include the glasslayer 82. The various types of heaters described above may be employedwith any of the embodiments described in FIG. 7, 8 or 11.

Referring now to FIG. 12, a random access PCR analyzer 120 isillustrated. In this embodiment, samples in sample vessels 122 and 124are provided to the DNA/RNA sample prep block 126. A sample preparationkit 130 may be provided with the thermal cycling devices. If required, areverse transcriptase reaction may be performed in block 128 to formcDNA from RNA. The sample preparation kit 130 is generally illustratedas block 130. The sample preparation kit 130 may be used to extract DNAmaterial from the input sample of tissue or fluid. The samplepreparation kit 130 may be integrated with the test kit. Afterpreparation the sample aliquots are taken from the samples and dispensedinto vessels A3, A2 and A1. Various primers, probes, specially bufferedsolutions, polymerases and dNTP's are illustrated in block 132 and maybe provided within the test kits 136, 138 and 140. During the PCRprocess the enzymes of the process have a reaction time. The reactiontime will vary depending on the particular process and the thermalabsorption rate of the sample. The circulation rate of the fluid is thuslimited by the absorption and the reaction time of the enzymes. Thermalcycling elements 142, 144 and 146 may also be provided within the testkits. Platens 148 and 150 may be used to provide the appropriatedenaturing and annealing temperatures to the thermal cycling elements.Prior to the insertion of the samples, the thermal cycling elements areinserted between the platens 148, 150, which form an incubator 152. Thethermal cycling elements 142, 144, 146 are thus brought up to thedesired temperature and the samples are drawn or forced through thedevice at a predetermined rate. Blocks 154 provide real-time detectionusing fluorescent excitation, lasers or the like. At various pointswithin the thermal cycling elements, the amount of amplification may bedetected. End-point detection 156 may be provided at the end of theprocess. The sample may be used for various types of analysis. After theend-point detection 156, the sample and the reagent mixture may bedisposed of.

The small size, expected low cost and disposable nature of the thermalcycling elements make possible the creation of a random access PCRanalyzer. Prior to this embodiment, virtual all PCR reactions wereperformed as batch operations with the number of different reactionsrunning simultaneously determined by the number of thermal cycling ovensin the system. One type of amplification system has two thermal cyclersand therefore can perform no more than two different protocolssimultaneously. As the number of PCR tests increases over time, therewill be more demand for random access analyzers. The thermal cyclingdevice may be capable of performing a number of tests. Random accessanalyzers may be defined as those having the ability to run anycombination of available tests on each and every sample presented to theanalyzer. Thus, various numbers of heaters or built-in heaters may beprovided for each thermal cycling element. Thus, various numbers ofsamples and various numbers of thermal cycling elements may besimultaneously performed. For example, the amount of heating may becontrolled by a central controller so that various temperatures may beachieved in various thermal cycling elements. Random access analyzershave several advantages over batch analyzers. That is, they are faster,more flexible, more versatile, and more productive than batch analyzers.It is envisioned that various PCR kits may be created each containing amultiplicity of thermal cycling elements of a specific type and thenecessary reagents to perform PCR amplification. The kits may includethe various polymerase enzymes, dNTP's, specially formulated buffers,and primers. Optionally, other agents for sample preparation anddetection may also be provided. Thus, by having the capability ofprogramming the temperatures, various PCR test protocol may be run. Theincubator 152 may include the means for drawing the fluid through thethermal cycling device. Also, various sample preparations may also beperformed such as lysing cellular samples to release DNA or performingreverse transcriptase reaction to create cDNA for RT-PCR.

Referring now to FIG. 13, a portion of the channel 54 is illustrated.This portion is illustrated as the first portion 58. A sample 160 isdisposed within the first portion of the channel 58. A pair of bubbles162, 164 delimit a sample 160 therein. The bubbles 162, 164 may beintermittently introduced into the flow with sufficient size to separatethe fluid into individual sample segments 160. This is done for thepurpose of promoting averaging of the temperature-time experience of theDNA solute through internal mixing (reducing the dispersion of dwelltimes) and segregation mixing also occurs as the fluid sample movesthrough a tortous path. The presence of the bubbles 162, 164 forcescirculation illustrated by the arrows 166 within the fluid segmentsample 160. Since the fluid at the wall must be stationary, there is aroughly toroidal flow profile. The spread in the velocity of any fluidelement and consequently the spread in its time temperature profile,will be limited by the extent of the segment. Practically, it may bedesirable to keep the ratio of bubbles to fluid as low as possible. Inthe figure, the ratio is about 20%. Since the figure also represents theoverhead associated with increasing the length of the fluid column, itis desirable to keep the ratio to a minimum. A measure of the maximumdispersion of times can be obtained by comparing the length of the fluidsegment to the length of a single cycle. If the fluid segment is 5-10diameters and the length of a single cycle is 100 diameters, thedispersion times would be about 5-10%. The use of the bubbles mayimprove the thermal cycling because the PCR devices generally operate atlow Reynolds numbers of less than 10 and often less than 1. This occursbecause the amount of fluid processed is small, in the range of severalnanoliters to tens of microliters. This also occurs because the heattransfer to the fluid is governed by its thermal relaxation content,which is proportional to the square of the depth and inverselyproportional to the diffusivity of the fluid. The time associatedtherewith is a few tenths of a second when the depths are between 100and 200 microns but may increase rapidly for channels of larger crosssection due to the squared dependence. When low Reynolds numbers areassociated with flow, the inertial effects are dominated by viscousforces and mixing is minimal or non-existent. In long channels flowapproaches the Poiseuille flow. In the Poiseuille flow situation, thereis no mixing. Material near the center flows at twice the averagevelocity while that near the walls creeps much more slowly. Thus, in aPCR environment the material near the center of the channel would racethrough the microchannels in about half the average time, potentiallylimiting the effectiveness of the cycle amplification. Material nearestthe wall on the other hand would move much slower than the average timebut other deleterious effects may be present. It should be noted thatthere is Brownian motion at the molecular level and thus some mixingdoes occur. Thus, by providing the bubbles 162, 164 described above,more thorough mixing may be provided.

Referring now to FIG. 14, a multiplicity of LED's 168 and photodiodes170 may be provided at various points or within various cycle portionsthroughout the process. As illustrated, LED's 168 and photodiodes 170may be provided in the first portion 58. Also, the location of thephotodiodes and LED's may be positioned in various locations in thecontinuous channels such as in each first portion, second portion orthird portion or various combinations thereof. For example, all or amultiplicity of the annealing paths may include an LED and photodiode.Light filtration elements may also be used to improve the detectionresults. The photodiodes may thus be coupled to an analyzer device formonitoring the amount of DNA present. As mentioned above, the amount ofDNA present may also be measured by fluorescence emissions through awindow. A laser or LED may provide the source for fluorescence to bemeasured. Wires 169, 171 may be coupled to a controller or analyzerdevice.

External means may also be used to allow recirculation of the PCRamplification product repeatedly through the same or separate devices.Between each of the devices a detection may be performed to determinethe amplification.

Referring now to FIG. 15, one example of a recirculating device coupledto a thermal cycling element 185 is illustrated. In FIG. 15 a pump 176is coupled to an upper chamber 178. That is, the pump 176 draws thesample into the chamber 178. To perform this, valve 180 is opened whichprovides the sample to the chamber 178 when the pump is aspirated.Valves 182 and 184 are closed. Then, valve 180 is closed, valve 182 isclosed, valve 184 is opened to provide venting, and the pump 176 isoperated to dispense the sample into the lower chamber 186. Thus, thethermal cycling element 185 amplifies the sample as it passes from thefirst chamber 178 to the second chamber 186. A recycle operation may beperformed by closing the first valve 180, opening the second valve 182,and opening the third valve 184 for venting. After the desired number ofcycles through the thermal cycling element 185 is performed, the fluidmay be dispensed by opening the first valve 180, opening the secondvalve 182, closing the third valve 184, and operating the pump in adispense mode. At various times a small portion may be dispensed or anamount of amplification may be measured in the thermal cycling element185.

As mentioned above, various techniques may be used for fabricating thedevice. For example, tubes may be preconfigured as set forth in FIG. 4and then a plastic material may be molded therearound. Insulation may bemolded into the various layers to separate the layers. Also, thelocations of the channel layers with respect to the exterior surfacesare chosen such that the channel pathways equilibrate at three desiredtemperatures required by the PCR protocol.

A method for operating the present invention includes introducing asample into the device having a plurality of layers. Prior to theintroduction of a sample, the temperatures of the individual layersmaintain three temperatures required in the PCR protocol. In thisembodiment, the temperature of the bottom thermally conductive layer islower than the upper thermally conductive layer. Thus, once a steadystate has been reached by the three conductive layers the sample isintroduced into the device. It should be noted that when thermalequilibrium, the entire upper layer is at the same temperature as thetemperature source and the entire lower layer is at the temperature ofthe lower temperature source. Thus, the device is in equilibrium. Themiddle layer is at an equilibrium temperature between the upper andlower temperatures. The sample is placed through the input port and inthe case of the second embodiment, is provided to an initial denaturingstage longer than the other denaturing stages. This may or may not berequired depending on the type of PCR performed. The PCR sample iscycled through the first portion of a cycle at the first temperature.The sample is moved to the second layer, which is the lower layerthrough the third layer in between the first and second layers. Thesample also passes through the insulators 24 and 26. Various means maybe used to force the fluid through the fluidic device such as a vacuum,a pressure pump, a capillary force or other means. The rate of fluidflow determines the length of time the PCR sample is subjected to thehighest temperature in the first portion. After the end of the firstportion is performed, the fluid passes to the bottom fluidic layerthrough the middle layer. After traversing all three channel portions ofthe first cycle, the fluidic sample enters the second cycle in the firstlayer. As is illustrated in FIG. 5, the various rows of first portionsmay be provided in a compact structure so more thermal cycles may beprovided in a small area.

It should be noted that various numbers of cycles may be provided invarious devices depending on the type of PCR to be performed. Greater orfewer number of cycles may be provided when compared to the 30 PCRcycles of FIG. 5. As mentioned above with respect to FIG. 15, thethermal cycling element may be coupled to a device for recirculating thefluid back into the device. This may be done as a feedback response todetection stage that detects the amount of amplification. If the desiredamount of amplification is not performed, the fluid sample may berecycled back into the thermal device. It should also be noted thatvarious reagents may also be input together with the sample at varioustimes so that a sufficient amount of reagents are available for thereaction.

Referring now to FIG. 16, a sixth embodiment of the present invention isillustrated. In this embodiment, a third temperature source 200 isillustrated. The third temperature source 200 includes a first contact202 and a second contact 204. The contacts may be configured in asimilar manner to that described above with respect to FIG. 11. That is,joule heating may be performed through the device and through the secondlayer 20 ^(v). That is, the extension layer may also be heated. In thisembodiment, the insulating layers 24 ^(v) and 26 ^(v) may have increasedinsulating properties so that each of the layers are or are nearlythermally independent. That is, in an embodiment with three temperaturesources it may be desirable to reduce or eliminate the thermaldependency of each of the layers. Of course, the heating may also beperformed by an independent heating layer such as a polysilicon heaterillustrated above. That is, an independent heater such as a polysiliconheater would be located between one or both of the insulating layers 24^(v), 26 ^(v), and the second layer 20 ^(v). Also, in this embodiment,various configurations for the first and second temperature sources maybe included. That is, various types of heaters may be included such as apolysilicon heater, surface heating or external platens may be employedto heat the device.

Referring now to FIG. 17, a seventh embodiment 210 of the presentinvention is illustrated. This embodiment has only two fluidic thermallyconductive layers rather than three as set forth in the previousembodiments. In this embodiment, a first layer 212 and a second layer216 are separated by an insulating layer 214. Layers 212 and 216 arethermally conductive layers. In this example, the first layer and thesecond layer have continuous channel 218 therein. The continuous channel218 has a first portion 220 and a second portion 222. The first portion220 is disposed within the first layer and the second portion 222 isdisposed within the second layer 216. In some forms of PCR, theextension temperature and the annealing temperature may overlap.Therefore, annealing and extension may be performed within the samelayer. Thus, this embodiment has two layers and two differenttemperature portions which form the cycle. It should also be noted thatthe insulating layer 214 is optional. That is, the insulating layer 214may be eliminated in some embodiments if a proper thermal gradient maybe applied to a device so that the first portion and second portion havethe desired temperature for the annealing and extension as well as thedenaturing layer.

This embodiment also may include the various means of heating describedabove. That is, various platens or other types of heaters may bedisposed at various portions of the device. Heaters may be disposedbetween the insulating layer 214 and the layer 216 or the layer 212, orboth. It should also be noted that various numbers of cycles may beprovided in the device.

Various embodiments with various numbers of cycles have beenillustrated. It should be noted that after or during a cycle variationssuch as detections or additional denaturing, annealing or extensionsteps may be performed. For example, an extra denaturing step after thelast extension step may be included. Also, elongated denaturing step maybe provided at the first cycle portion or first and second cycleportions.

Referring now to FIGS. 18 and 19, a test fixture 250 for a thermalcycling device 252 is illustrated. At the center of the thermal cyclingdevice 252 is a fluidic device 12. The thermal cycling device 252includes the fluidic device 12 and includes an upper cap assembly 254and a lower cap assembly 256. A fixture assembly 258 includes legs 260coupled to a mounting top 262. The mounting top 262 includes recessedportions 264 for receiving the thermal cycling device 252. The upper capassembly 254 and lower cap assembly 256 are a denaturing heater and anannealing heater in a PCR device.

The upper cap assembly includes a cap housing 270 having a channel 272therein. The channel 272 receives a platen 274. An end cap 276 is usedto secure the platen within the cap piece 270. A rod heater 278 andthermocouple 280 are inserted within an opening 282 in end cap 276. Therod heater is used to heat the platen to a predetermined temperature sothat the temperature is imposed upon the fluidic device 12. Morespecifically, the upper surface of the fluidic device 12 is coupled tothe platen 274.

Lower cap assembly 256 includes a lower cap housing 290 that includes aguide feature 292 to be received within the recesses 264 of top 262. Aplaten 294 is received within the lower cap assembly. A rod heater 296and a thermocouple 298 are inserted within an opening 300 in an end cap302 so that the rod heater provides heat to the lower platen 294 andthermocouple 298 is used to heat the platen to a predeterminedtemperature. Platen 294 may also be coupled to an input tube 310 and anoutput tube 312. An input channel 314 and an output channel 316 may beprovided through the platen. Gaskets 318, 320 may be coupled between thefluidic device 12 and the platen 294 to prevent fluid leakagetherebetween. The device is held together with threaded fasteners 340.

A heat sink 350 may be coupled to the lower cap 290. Because the upperplaten 274 is at a higher temperature than the lower platen 294, heat isdissipated through the heat sink 350. This helps maintain the denaturinglayers and annealing layers of the fluidic device 12 at thepredetermined temperatures.

A pair of hose clamps 352, 354 may also be used to secure the input tube310 and output tube 312 to the thermal cycling device 252.

In one constructed embodiment of the fluidic device, a cross-sectionalchannel area of 0.022 mm² was used. The channel length for thedenaturing portions was 1 mm and the extension portions 2 mm. Thirtycycles were used in the constructed embodiment. The preliminarydenaturation channel length was 9 mm. The thickness of the siliconlayers was 0.575 mm. The conductivity was 130 watts/m° C. The polyimidethickness was 0.125 mm and its conductivity was 0.31 watts/m° C.

The reaction solution may include human genomic template DNA of 5 femtomolar concentration. A commercial PCR buffer having criticalconstituents of tris-HCl buffer for pH stability and MgCl₂ to provideMg⁺⁺ ions critical for polymerase activity. Deoxynucleotidetriphosphates (dNTP) were provided of 200 micro molar concentrationseach for A, T, C and G. Forward and reverse primers specific to thehuman genome template which are 20-30 nucleotides in length were chosento define 100-200 base pair amplicons of 200 micro molar concentrationseach. Taq polymerase enzyme was chosen at one unit per reaction. Thetest fixture described above is coupled to a syringe pump (not shown)from which the reaction solution is pumped through the thermal cyclingdevice 12. The heated platens maintain the denaturation temperatures of96±1° C. and annealing temperatures of 54±1° C. The platen heaters andtemperature controllers having embedded thermocouples provide 16 wattseach. A heat sink having a thermal resistance of 3.75° C./w wereattached to the exposed surface of the annealing platen. The syringepumps that were used have a capacity of 10 μL and flow rate of 0.22 μL/swith a 1 mm/s flow rate. The amplification product is collected at theoutput of the device. Various means may be used to detect the presenceand the amount of amplified DNA material present in the product. Commonmethods employ electrophoresis to spatially separate the PCR products bymolecular weight and fluorescence probes that bind to the DNA product toindicate the concentrations.

FIG. 20 schematically illustrates a three-temperature device 400 inwhich some of the fluid channels (i.e. “channel segments”) are formed inthe insulating layers. The device 400 includes a first thermalconducting (denaturation) layer 402, a second thermal conducting(annealing) layer 404, and a third thermal conducting (extension) layer406. The three layers are separated by insulating layers 408 and 410.The cycling channel 412 includes channel segments 414, 416, 418, and 420which reside in the insulating layers. It is preferred, but notessential, that the channel segments in the insulating layer be formedso that one side of the channel segments includes one of the thermalconducting layers. This allows the fluids in the channel segments tohave direct contact with the conducting layer that is at the desiredtemperature. The insulating layer channels are “adjacent” the conductinglayers whether or not they are in direct contact with the conductinglayer.

Forming channel segments in the insulating layer is easier and lessexpensive than forming the channel segments in the conducting layers.The insulating layers are typically made from a plastic or polymericmaterial and the channel segments can be easily formed in them byinjection molding techniques. This eliminates the expensive etching orother processes described above which can be used to form the channelsin the conducting layers.

FIGS. 21A and 21B illustrate a two-layer thermal cycling device 430which has channel segments in the insulating layer. The device has afirst conducting (denaturation) layer 432, a second conducting(annealing/extension) layer 434, and an insulating layer 436. The layer432 is held at the denaturation temperature, while the layer 434 is heldat the combined annealing extension temperature. The fluid path in thedevice 430 alternatives from left to right, as shown in FIG. 21A andthen from right to left as shown in FIG. 21B.

The device shown in FIG. 20 also is a “folded channel” device, that isthe extension channel segments exist in two planes on alternate sides ofthe conducting layer 406. This same principle can be applied to make thethermal cycling device smaller since it is often desirable to have theextension layer much longer than the denaturation and annealing layers.By using both sides of the extension layer, the device can be made morecompact.

It is also possible to have the channel “fold back” and provide two ormore channel segments in either the annealing or denaturing layer.Similar savings in size and cost could be realized. In addition, therecould be multiple folds in any of the layers making the device morecompact.

FIGS. 20, 22 and 23 illustrate three devices using the “folding channel”principle. In FIG. 20, the device 400, which has three conductinglayers, has a folded extension channel, with part of it residing ininsulating layer 408 and the other part in insulating layer 410. In FIG.22, the device 440, which has three conducting layers, again has afolded extension layer, with part of it residing in the upper face ofconducting layer 443 and the other part in the lower face of conductinglayer 443. In FIG. 23, the device 450, which has two conducting layers,has a folding annealing-extension channel, with part of it residingwithin conducting layer 452 and part of it residing on the top surfaceof conducting layer 452.

In the multiple layer devices described above, the layers could be ofthe same or dissimilar materials. FIGS. 24 and 25A/25B depict twodevices in which the layers are made of homogeneous materials.

In FIG. 24, the device 460 is a three temperature device usinghomogeneous materials. The external platens 461 and 462 take the placeof the conducting layers 441 and 442 which are present in FIG. 22. Thelayers 1 and 4 (461 and 462) are sufficiently thin such that there isonly a negligible temperature drop across them.

FIGS. 25A and 25B depict a two-temperature device 470 using homogenousmaterials. Consecutive cycles are shown in FIGS. 25A and 25B. Layers 1(471) and 3 (472) are sufficiently thin such that there is a negligibletemperature drop across them.

FIGS. 26 and 27 depict a thermal cycling device 480, with FIG. 27 beingan exploded view thereof. The device 480 includes at least oneconducting layer 481 which has a pair of tabbed connectors 482A and482B. The tabbed connectors are used to connect the thermal heating andheat sinking sources to the conducting layer 481. Layer 481 is aconducting denaturation layer and is equipped with tabbed connectors(“tabs”) that provide a large surface area over which to conduct heat.

FIG. 28 illustrates a two-temperature device 490 which has a clear coverwindow (glass layer 491) over the denaturation layer 402. The clearwindow layer allows real time PCR with detection on theannealing/extension side of the thermal cycling device 490. Withtwo-temperature devices, real time detection during annealing/extensioncould be advantageous because more channel area 493 is exposed, as shownin FIG. 28.

While the invention has been described in connection with one or moreembodiments, it should be understood that the invention is not limitedto those embodiments. On the contrary, the invention is intended tocover all alternatives, modifications, and equivalents, as may beincluded within the spirit and scope of the appended claims.

What is claimed is:
 1. A three-layer apparatus for performing apolymerase chain reaction comprising: a first thermally conductivelayer; a second thermally conductive layer; a third thermally conductivelayer; a first insulating layer positioned between said first and secondthermally conductive layers; a second insulating layer positionedbetween said second and third thermally conductive layer; and acontinuous channel having channel segments formed within at least one ofsaid insulating layers, and at least one of said thermally conductinglayers; said first, second and third thermally conductive layers beingpositioned one on top of the other forming a multi-layer apparatus. 2.The three-layer apparatus as described in claim 1 wherein one of saidchannel segments in said first insulating layer is adjacent to saidfirst thermally conductive layer and one of said channel segments insaid second insulating layer is adjacent to said third thermallyconductive layer.
 3. The three-layer apparatus as described in claim 1wherein said first thermally conductive layer is a denaturing layer,said third thermally conductive layer is an annealing layer, and saidsecond thermally conductive layer is an extension layer.
 4. Thethree-layer apparatus as described in claim 3 wherein at least one ofsaid channel segments extends for a first direction in its respectivelayer and then reverses itself and extends in the opposite direction inthe same layer before proceeding to another layer.
 5. The three-layerapparatus as described in claim 4 wherein said at least one of saidchannel segments extend for said first direction on one side of itsrespective layer and then reverses itself and extends in said oppositedirection on the opposite side of said same layer before proceeding toanother layer.
 6. The three-layer apparatus as described in claim 5wherein said respective layer is an extension layer and said at leastone of said channel segments is at least partially formed in aninsulating layer.
 7. The three-layer apparatus as described in claim 1further comprising at least one temperature source for maintaining saidfirst thermally conductive layer at a first temperature and formaintaining said third thermally conductive layer at a secondtemperature lower than said first temperature.
 8. A two-layer apparatusfor performing a polymerase chain reaction comprising: a firstconducting layer; a second conducting layer; an insulating layerpositioned between said first and second conducting layers; a continuouschannel having a plurality channel segments, at least one of saidchannel segments positioned in said insulating layer wherein said firstconducting layer is positioned on top of said second conducting layer.9. The two-layer apparatus as described in claim 8 wherein at least onechannel segment is adjacent to said first conducting layer and at leastone channel segment is positioned adjacent to said second conductinglayer.
 10. The two-layer apparatus as described in claim 8 furthercomprising at least one temperature source for maintaining said firstand second conducting layers at prespecified temperatures.
 11. Thetwo-layer apparatus as described in claim 10 wherein one of saidconducting layers has a tab member thereon for connecting to saidtemperature source.
 12. The two-layer apparatus as described in claim 8wherein at least one channel segment is a denaturing segment, and atleast one channel segment is an annealing-extension segment.
 13. Thetwo-layer apparatus as described in claim 12 wherein at least twochannel segments extend for a first direction in one of said layers andthen reverse themselves and extend in the opposite direction in saidsame layer.
 14. The two-layer apparatus as described in claim 12 whereinat least two annealing or denaturing segments are present in the samelayer or adjacent the same layer.
 15. A method for performing apolymerase chain reaction comprising: (a) introducing a sample into athree-layer apparatus, said three-layer apparatus comprising: a firstthermally conductive layer; a second thermally conductive layer; a thirdthermally conductive layer; said first, second and third thermallyconductive layers being positioned one on top of the other forming athree-layer apparatus; a first insulating layer positioned between saidfirst and second thermally conductive layers; a second insulating layerpositioned between said second and third thermally conductive layer; andat least one temperature source for maintaining said first thermallyconductive layer at a first temperature and for maintaining said thirdthermally conductive layer at a second temperature lower than said firsttemperature; a continuous channel having channel segments formed withinat least one of said first insulating layers and at least one of saidthermally conducting layers; (b) performing a first portion of a cyclein a first channel segment at a first temperature; (c) moving the sampleto another layer; (d) thereafter, performing a second portion of thecycle in a second channel segment at a second temperature; (e) movingthe sample to a second channel segment; (f) thereafter, performing athird portion of a cycle in a third channel segment at a thirdtemperature; and (g) repeatedly performing the first portion, the secondportion and the third portion for a predetermined number of cycles toperform the reaction.
 16. A method for performing a polymerase chainreaction comprising: (a) introducing a sample into a two-layer apparatuscomprising: a first conducting layer; a second conducting layer; saidfirst and second conducting layer being positioned one on top of theother forming a two layer apparatus; an insulating layer positionedbetween said first and second conducting layers; and a continuouschannel having at least one channel segment within said insulatinglayer; (b) performing a first portion of a cycle in a first channelsegment at a first temperature; (c) moving the sample to a secondchannel segment; (d) thereafter, performing a second portion of thecycle in said second channel segment at a second temperature; (e) movingthe sample to a third channel segment; (f) thereafter, performing athird portion of a cycle in said third channel segment at a thirdtemperature; and repeatedly performing the first portion, the secondportion and the third portion for a predetermined number of cycles toperform the reaction.
 17. A multi-layer apparatus for performing apolymerase chain reaction comprising: a first thermally conductivelayer; a second thermally conductive layer; a third thermally conductivelayer; a first insulating layer positioned between said first and secondthermally conductive layers; a second insulating layer positionedbetween said second and third thermally conductive layers; a first heatsource for maintaining said first thermally conductive layer at a firsttemperature; a second heat source for maintaining said third thermallyconductive layer at a second temperature lower than said firsttemperature; said first, second and third thermally conductive layersbeing positioned one on top of the other forming a multi-layerapparatus; and a continuous channel formed in said multi-layer apparatusfor performing a polymerase chain reaction, said channel having a firstchannel segment in said first insulating layer adjacent said firstthermally conductive layer, and a second channel segment in said firstinsulating layer adjacent said second thermally conductive layer, athird channel segment in said second insulting layer adjacent saidsecond thermally conductive layer, and a fourth channel segment in saidsecond insulting layer adjacent said third thermally conductive layer.18. A two-layer apparatus for performing a polymerase chain reactioncomprising: a first thermally conductive layer; a second thermallyconductive layer; an insulating layer positioned between said first andsecond thermally conductive layers; said first and second thermallyconductive layers being substantially the same size and being positionedone on top of the other forming a multi-layer apparatus; and acontinuous channel formed in said multi-layer apparatus for performing apolymerase chain reaction, said channel having at least one of thechannel segments positioned in the insulating layer.
 19. A two-layerapparatus as described in claim 18 wherein all of the channel segmentsare positioned in the insulating layer.
 20. A multi-layer PCR device forprocessing a nucleic acid sample comprising: at least two thermallyconductive layers; at least one insulating layer; said thermallyconductive layers and said insulating layer and being positioned one ontop of the other forming a multi-layer device; and a continuous channelin said multi-layer device for circulation and processing of a nucleicacid sample.
 21. The device as described in claim 20 further comprisingheating sources for heating said thermally conductive layers.
 22. Thedevice as described in claim 20 wherein said continuous channelcomprises a plurality of channel segments and wherein at least onechannel segment is located in said insulating layer.
 23. The device asdescribed in claim 22 wherein all of said channel segments are locatedin said insulating layer.
 24. The device as described in claim 20wherein three thermally conductive layers are provided.
 25. The deviceas described in claim 20 wherein two insulating layers are provided. 26.The device as described in claim 20 wherein three thermally conductivelayers and two insulating layers are provided.
 27. The device asdescribed in claim 26 wherein said continuous channel has a plurality ofchannel segments and at least one channel segment is located in one ofsaid insulating layers.
 28. The device as described in claim 26 whereinsaid continuous channel has a plurality of channel segments and at leastone channel segment is located in each of said insulating layers. 29.The device as described in claim 26 wherein said continuous channel hasa plurality of channel segments and a plurality of said channel segmentsare located in said insulating layers.
 30. The device as described inclaim 26 wherein each of said two insulating layers is positionedbetween two of said thermally conductive layers.
 31. The device asdescribed in claim 20 wherein one insulating layer and two thermallyconductive layers are provided, and wherein said insulating layer ispositioned between said two thermally conductive layers.
 32. The deviceas described in claim 31 wherein said continuous channel comprises aplurality of channel segments and wherein one of said channel segmentsis located in said insulating layer adjacent to one of said thermallyconductive layers.
 33. The device as described in claim 26 wherein saidfirst thermally conductive layer is a denaturing layer, said thirdthermally conductive layer is an annealing layer, and said secondthermally conductive layer is an extension layer.
 34. The device asdescribed in claim 22 wherein at least one of said channel segmentsextends for a first direction in its respective layer and then reversesitself and extends in the opposite direction in the same layer beforeproceeding to another layer.
 35. The device as described in claim 30wherein said continuous channel comprises a plurality of channelsegments and wherein one of said channel segments is located in saidfirst insulating layer adjacent to said first thermally conductive layerand one of said channel segments is located in said second insulatinglayer adjacent to said third thermally conductive layer.
 36. The deviceas described in claim 30 wherein said first thermally conductive layeris a denaturing layer, said third thermally conductive layer is anannealing layer, and said second thermally conductive layer is anextension layer.
 37. The device as described in claim 35 wherein atleast one of said channel segments extends for a first direction in itsrespective layer and then reverses itself and extends in the oppositedirection in the same layer before proceeding to another layer.